Accepted Manuscript

Utilization of agro-wastes to inhibit aflatoxins synthesis by Aspergillus para-

siticus: A biotreatment of three cereals for safe long-term storage

B. Sultana, R. Naseer, Poonam Nigam

PII: S0960-8524(15)01219-5
DOI: http://dx.doi.org/10.1016/j.biortech.2015.08.113
Reference: BITE 15466

To appear in: Bioresource Technology

Received Date: 18 May 2015

Revised Date: 17 August 2015
Accepted Date: 18 August 2015

Please cite this article as: Sultana, B., Naseer, R., Nigam, P., Utilization of agro-wastes to inhibit aflatoxins synthesis
by Aspergillus parasiticus: A biotreatment of three cereals for safe long-term storage, Bioresource Technology
(2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.08.113

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Utilization of agro-wastes to inhibit aflatoxins synthesis by Aspergillus parasiticus: A
biotreatment of three cereals for safe long-term storage

B. Sultanaa, R. Naseerb, Poonam Nigamc*

a Department of Chemistry, University of Agriculture, Faisalabad, Pakistan

b Department of Chemistry, Government College Women University, Faisalabad, Pakistan
c Faculty of Life & Health Sciences, University of Ulster, Coleraine, Northern Ireland, UK

*Corresponding author.
E-mail address: p.singh@ulster.ac.uk (P. Nigam)

Abstract
The growth of Aspergillus parasiticus and aflatoxins production were inhibited during
storage of three important cereals (wheat, maize and rice) using leaves of neem (Azadirachta
indica) and kikar (Acacia nilotica). Cereals were inoculated with mould spores and stabilized by
neem and kikar leaves-powder. Test samples with moisture levels of 21% were stored at 30 oC
for a period of 9 months. Aflatoxins were quantified at different time intervals in stored cereals.
Neem leaves fully inhibited all types of aflatoxins synthesis for 4 months in wheat and for 2
months in maize while in rice inhibited synthesis of only B2, G1 and G2 aflatoxin for 3 months.
Kikar leaves fully inhibited aflatoxin B2, G1 and G2 for 3 months in wheat, and for 2 months in
maize. Among two investigated plants, neem leaves were found more effective for preventing
the production of all types of aflatoxins in cereals’ long-term storage.

Keywords: A. parasiticus; aflatoxins; cereals; neem; kikar

1. Introduction
Aspergillus species are most dominant among food deteriorating moulds. They widely
occur in food particularly in starchy cereal grains such as wheat, maize, sorghum, rice, barley
and millet and may produce toxic secondary metabolites called aflatoxins (El-Aziz et al., 2012).
Among the 20 identified aflatoxins, B1 and B2 are produced by Aspergillus flavus, while
aflatoxin G1 and G2 along with B1 and B2 are produced by Aspergillus parasiticus (Bennett and
Klich, 2003). Chemically aflatoxins are polycyclic difuranocoumarins compounds in which
bifuran group is attached at one side of coumarin nucleus and a pentenone ring attached to other

1
side in case of aflatoxin B series, or a six-membered lactone ring in case of aflatoxin G series
(Nakai et al., 2008). They are highly stable at wide range of temperature being thus persistent in
contaminated plants during the processing, cooking and sterilization (Sirot et al., 2013).
Aflatoxins have been classified as carcinogenic in humans by the International Agency for
Research on Cancer (IARC, 2002) and it has been proved from in vitro studies that aflatoxins are
causative agent for liver cancer (Chung et al., 2009; Lui and Wu, 2010). Due to their metabolic
disorders in humans, mycotoxins have been also classified as mutagenic, immunosuppressant
and carcinogenic (Wild & Gong, 2010).
Cereal grains are an important commodity both for humans as well as for animals due to
their high contents of proteins, carbohydrates and fibers and low price. However, these are very
sensitive to mycotoxins contamination (Murphy et al., 2006). Cereal grains may be contaminated
at any stage in field, during storage or processing, by moulds capable of producing mycotoxins
(Paterson and Lima, 2010). In European Union, 20% of the crops production is contaminated by
moulds and their toxic metabolites present a serious threat to human and animals health (Murphy
et al., 2006; Naseer et al., 2014).
This is an alarming situation particularly for Asians as their diet mainly consists of
cereals. The situation is further affected by the warmer climate of Asian countries, which favors
mould growth and aflatoxins synthesis. Furthermore, harvesting season in Asian countries
usually fall at the start of the rainy season and sun drying of cereals cannot be possible for a
farmer, which is the common practice applied in these areas. Thus, cereal-grains with high
moisture level enters the storage system. As a result one quarter of food crop of the world,
including common crops like rice, maize and wheat are infected by mycotoxins contamination
(Ahsan et al., 2010).
During last ten years, there was no prominent development in the use of safe and novel
antimicrobial drugs for the storage of cereals while the microbial resistance problems have
astronomically grown (Mathews et al., 2011). Certain phytochemicals have been identified with
high potency antimicrobial attributes (Abichandani et al., 2010; Chitnis et al., 2007), but many of
them cannot be used safely in foods (Hussain et al., 2010 a,b; Sauvage et al., 2010). A number of
plant extracts with efficient antifungal bioactivity have been screened that can be used to control
foodborne pathogens and can inhibit biosynthesis of mycotoxins (Granger et al., 2009; Genest et
al., 2008; Hussain et al., 2011a,b). Neem (Azadirachta indica) leaves extract have shown

2
inhibitory effects on the growth of different moulds including A. flavus, A. fumigatus, A. niger,
A. terreus, C. albicans and M. gypseum (Mahmoud et al., 2011). Due to potent antifungal
potential of neem leaves against A. parasiticus, they can be used to inhibit mould growth and the
biosynthesis of aflatoxins (Costa et al., 2010). Fungicidal and antiaflatoxigenic properties of
neem leaves have been ascribed to numerous active constituents like desactylimbin, quercetin, β-
sitosterol, tetranotriterpenoids and volatile compounds (Choudary, 2002).
Acacia nilotica (kikar) contains variety of bioactive components such as alkaloids, phenolic acids,
tannins, terpenes and saponins which are responsible for substantial antimicrobial activity against
mould and bacteria (Banso and Adeyemo, 2007). Search of antifungal agents of plant origin is
one of the novel approaches for the replacement of carcinogenic synthetic preservatives.
However, data is yet insufficient regarding the direct applications of plant materials to protect
cereals from mould.
Therefore, the present research work was aimed to inhibit the growth of toxigenic mould,
Aspergillus parasiticus by using two native plants neem and kikar widely grown in Pakistan
which may preserve cereal grains from aflatoxins contamination during long-term storage.

2. Materials and Methods

2.1 Isolation, purification and identification of mould

A. parasiticus strain used in present study was isolated from contaminated corn and
wheat grains (3 samples for each) using agar plate method. Three plates for each were incubated
for seven days under darkness at 28 oC and developed spores were inoculated on PDA (potato
dextrose agar) (Oxoid, UK) medium containing antibiotic (streptomycin) by using hyphal tip or
single spore technique and cultivated at 28 oC. Isolated mould species were observed under
microscope and characterization of A. parasiticus species was based on morphological feature,
colony growth, organization of conidial heads (columnar or radiated) and the color of colonies.
The mould isolates having general characteristic of A. parasiticus were transferred to the PDA
slants, grown and preserved at 4 oC.
The toxigenicity of the isolates was determined by observing the fluorescence (green and
blue color) of the PDA medium under UV lamp. Isolation, purification and identification were

3
performed in Department of Mycology and Plant Pathology, University of Agriculture,
Faisalabad.

2.2 Preparation of inoculum

The inoculum of A. paraciticus was prepared in PDA (Oxoid, UK) at 28 oC and after well
sporulation (usually in 7 days), spores were washed out using sterilized water with 0.1% Tween
80. The required concentration 1012 spores/mL was adjusted by adding water and counting in
haemocytometer. Spore suspension was prepared one day before use and preserved at 4 oC.

2.3 Inhibitory potential of plant materials on aflatoxins production in cereals (maize, wheat and
rice)
2.3.1 Collection of plant Samples and cereals
Neem and kikar leaves were collected from local agricultural resources. About 15 kg of
each kind of freshly harvested cereal grain samples i.e. maize (Zea mays) variety Moon Star,
wheat (Triticum aestivum L.) variety Sehar, and rice (Oryza sativa L.) variety Super Basmati
were obtained from Ayub Agricultural Research Institute, Faisalabad.
2.3.2 Moisture content and pretreatment
The moisture content of cereal grains (maize, wheat and rice) was estimated by drying
replicate portion of 5 g of grains. The grains were subsequently dried to a constant weight in an
oven at 105 oC to remove moisture and loss in weight was expressed as percentage calculated on
wet weight basis. The moisture content of dried maize, wheat and rice were found to be 11.10%,
9.76% and 10.78, respectively.
Three cereal grains were individually washed with tap water and dried in an oven at 40-
70 oC. Each lot was divided into 64 portions separately, each containing 200 g of grains of their
respective variety. Each portion was autoclaved separately to kill microorganisms, and packed in
air tight plastic bags aseptically under air laminar flow chamber. The grains were moistened with
sterilized distilled water to raise the required moisture level to 21% and inoculated by mixing 4
mL of spore suspension (1012 spores/mL) of A. parasiticus to each bag separately.

2.3.3 Stabilization of cereals with plant treatments

Neem and kikar leaves in powdered forms at three concentration of 5%, 10%, 20%, w/w
were added separately to each plastic bag containing 200 g of inoculated cereal and were shaken

4
thoroughly to mix plants powder uniformly with grains. Untreated grain sample (without leaves)
was used as control. The treated and control cereal samples with moisture level of 21% were
stored at 30 oC for a period of 9 months.
At different time intervals cereal samples were taken and analyzed by TLC (thin layer
chromatography) technique for the presence of aflatoxins, and these were quantified by HPLC
(high performance liquid chromatography) technique. The inhibitory effect of leaves on
aflatoxins synthesis was estimated from the difference in amount of aflatoxins contents produced
in treated samples as compared to their respective controls.

2.4 Analysis of aflatoxins in cereals.

2.4.1 Extraction of aflatoxins
Extraction of aflatoxins from cereal grains was made following a well-established
method of (Beltran et al., 2011) with some modifications. A ground sample (20 g) of
representative cereal grain was taken in a 250 mL conical flask and was extracted using mixture
(80 mL) of acetonitrile-water (84:16) in an orbital shaker at ambient temperature (average
temperature 37 oC) for 90 min. The extract was filtered through Whatman filter paper No. 4 and
filtrate was concentrated under reduced pressure at 50 oC to final volume of 2-5 mL.
2.4.2 Analysis of aflatoxins
The qualitative analysis of extracts for the presence of aflatoxins was done by TLC
technique using silica gel G60 plates (20×20 cm; Merck). The plates were dipped in solution of
oxalic acid in methanol (10% wt/wt) for 2 min and heated at 110 oC for 2 min and after cooling
the plates, 50 µL of sample was spotted (5 µL of extract dissolved in 1 mL of methanol). The
plates were developed using chloroform:acetone (9:1, v/v) as solvent system. Plates were dried
and observed under UV light (λ = 365nm) in an enclosed Camag 2930 UV visualizer (Germany).
The blue fluorescence showed the presence of aflatoxins B and green fluorescence indicated G
type aflatoxins.
The quantification of aflatoxins was done by HPLC, concentrated extract sample was
diluted with 20 mL of deionized water and to increase sensitivity and selectivity, sample was
passed through aflatest immunoaffinity column (Vicam, Romer Lab., USA) at a flow rate of 2
mL/min. The column was washed using 2 mL of deionized water and dried for 1-2 min by air

5
streaming. Then aflatoxins were eluted slowly using 2 mL methanol step wise (1 mL each) and
methanol was evaporated under N2 blanket.
Pre column derivitization of aflatoxins was done to increase detection and aflatoxins recovery.
Almost-dried sample of aflatoxins was vortexed with 200 µL of n- hexane for 30 sec to remove
fat; 50 µL of TFA (trifluoroacetic acid) was added in sample mixture and vortexed for 30 sec.
Then 1.95 µL water: acetonitrile (9:1) was added and vortexed for 20 sec; resultant 20 µL
sample was used for HPLC analysis.
Before analysis, the instrument was validated for its precision, accuracy and limit of
detection (LOD). Calibration curve was drawn using solutions of aflatoxins in acetonitrile at
concentration of 0.05, 0.1, 0.5, 1.0, 5.0, and 10 µg L-1. The separation (resolution) pattern of
aflatoxins was assayed by reverse phase HPLC column.
Aflatoxin contents were expressed in terms of ng/g, and aflatoxin inhibition was calculated using
formula;
Percentage of inhibition = [Y – X / Y] x 100
X” is the concentration of aflatoxin in treated sample and Y” is the concentration of aflatoxin in
control. The limit of detection (LOD) for each aflatoxin was 0.02 ng/g.

2.5 Statistical Analysis

All determinations were made in triplicate and the results of various parameters are described
as (n=3×3) ± SD (n=3×3). Moreover, interaction between two variables (cereal and plant
leaves) was also taken into consideration. For this purpose best fit statistical design
(Montgomery, 2000) i.e. two-way Analysis of Variance (ANOVA) was accomplished on all
measurements to find significant differences at significant level less than 5% (p<0.05) by using
Minitab 2000 version. Statistical software (Minitab Inc. Pennysalvania, USA)

3 Results and discussion

3.1 Fungal strains
The 30 developed colonies of different mould species were isolated from maize and
wheat samples belonging to genera Fusarium, Pencillium, Trichoderma, Aspergillius, Alternaria,
Rhizopus. Out of thirty different isolates 6 colonies of Aspergillus parasiticus were isolated and
2 isolates were found positive producer of aflatoxins.

6
3.2 Potential ability of test mould to produce aflatoxins in cereals
The aflatoxigenic strain of A. parasiticus was used due to the fact that A. parasiticus are
the most undesirable species of Aspergillus that are ubiquitous and can invade under favorable
conditions of temperature and moisture on variety of cereal grains. The presence of aflatoxins in
control cereal samples (without any additive) stored at 21% moisture and 30 oC temperature
showed the presence of B and G types of aflatoxins and by comparing the Rf values of tested
samples with the standards four type of aflatoxins i.e. B1, B2, G1 and G2 were identified. Our
findings that A. parasiticus was responsible for the production of four types of aflatoxins i.e. BI,
B2, G1 and G2 are in fair agreement with the investigations of Sharma and Sharma (2012) who
have reported the production of aflatoxin BI, B2, G1 and G2 by A. parasiticus.
Quantitative estimation by HPLC showed that the total aflatoxins contents
(B1+B2+G1+G2) accumulated by A. parasiticus in control samples of cereals were 29.64 ng/g in
rice, 19.06 ng/g in maize and 12.80 ng/g in wheat after one month of storage period. Results
indicated that rice is more susceptible to aflatoxins production followed by maize and wheat.
Among total aflatoxins contents accumulated in rice, maize and wheat; aflatoxin B1 was at
highest level of 19.12, 12.17 and 8.19 ng/g, respectively, and aflatoxin G1 was 6.88, 4.82 and
3.00 ng/g, respectively; while aflatoxin B2 was detected in small amount (2.72, 1.93 and 0.96,
respectively). However, very small amount of aflatoxin G2 was also detected at these storage
conditions (0.91, 0.11 and 0. 65ng/g, respectively). Accumulation of aflatoxins was in the order
of BI > G1> B2 > G2. Statistical analysis showed that the amount of aflatoxin B1 produced by
tested strain in three different cereals was found to be significantly p<0.05 higher than the other
three types of aflatoxins (B2, G1 and G2). The germination and growth of mould spores and
mycelia were affected by the type of starchy food and the availability of nutrients in form of
protein and starch.

3.3 Inhibitory effect of leaves on aflatoxins production during storage of cereals (maize, wheat
and rice)
Many countries have worked out the maximum permitted limit of aflatoxins according to
different consumers and commodities, in European countries, maize should not have more than
5.0 µg/g of aflatoxin BI level (Zou et al., 2012). Due to the adverse effects of synthetic

7
pesticides/fungicides on human health and environment, there is a need to search for natural and
eco-friendly antimicrobial (Hussain et al., 2011a, b) and fungicides with better performance
(Mohanlall and Odhav, 2006; Efstratiou et al., 2012; Hussain et al,. 2010b).
The results of two plant materials’ inhibitory effect against the production of aflatoxins
by A. parasiticus in three cereals are presented in Tables 1-6. Neem and kikar leaves at three
different concentrations (5, 10, and 20%) were applied to three cereals to investigate their
inhibitory effect on aflatoxin (B1, B2, G1 and G2) production by A. parasiticus. The inhibitory
effect of leaves was estimated by comparing aflatoxins contents produced in control ceral-
esamples (without any leaf-additive) with that produced in leaf-treated samples. Generally the
production of aflatoxins in treated samples was found to be less as compared to control samples
during 9 months of storage. Although both plant treatments at all concentration inhibited the
synthesis of aflatoxins in cereals; however, 20 % was found most effective. In general, a direct
relationship was observed between the applied plant concentration and their inhibitory effect on
aflatoxins production.
In cereal wheat neem leaves were found more effective at 20% concentration which
completely inhibited the synthesis of aflatoxin B1 for 4 months, G1 for five months and B2 and
G2 synthesis for seven months (Table 1), 10% concentration was also effective that fully
inhibited G1, B2 for four months and G2 for seven month while 5% was found least effective.
Whereas, kikar leaves at 20% completely inhibited aflatoxin B2 and G1 synthesis for 3 months
and G2 for seven months (Table 2) and 10 % fully inhibit only G2 synthesis for seven months.
In maize (Table 3) neem leaves at 20% concentration fully inhibited the synthesis of
aflatoxin B1 for 2 month, G1 for 3 months and B2 and G2 for four months. While kikar leaves at
20% concentration fully inhibited only the synthesis of aflatoxin B2, G1 for one month and G2 for
five months (Table 4).
In rice neem leaves (Table 5), at 20% concentration fully inhibited the synthesis of
aflatoxin B2 for 3 months, G1, for four months and G2 for five months but for aflatoxin B1
inhibitory effect was 96 % during 1st month of storage. While inhibitory effect of kikar leaves at
20% concentration was found 73%, 86% and 92% for aflatoxin B1, B2, and G1 synthesis,
respectively during 1st month of storage (Table 6).
In our study, the pattern of a significant decrease in aflatoxins contents by increasing the
plants concentrations was same as observed by Sharma and Sharma (2012) in stored maize, who

8
have also reported that by increasing the concentrations of extracts in stored maize, a rapid
decrease in aflatoxins amount was found. Significant differences (p<0.05) were observed in
aflatoxins amounts produced by A. parasiticus in control and plant treated cereal samples.
It is evident from results that for wheat and maize, in addition to 20%; 5 and 10%
concentrations of neem leaves also showed good inhibitory potential for aflatoxin B1, B2, G1 and
G2 accumulation. The amount of aflatoxins in wheat was found lower than other two cereal
grains (rice and maize), confirming that both tested plants showed higher inhibitory potential
against mould growing on wheat than other two cereals. Statistical analysis showed that a
significant difference was observed among the inhibitory effects of plants against aflatoxins
production by A. parasiticus in three cereals.
According to our results at initial stages of storage period both tested plants fully
inhibited the aflatoxins synthesis for specific time period but after this period aflatoxins contents
were detected up to 9th month. The presence of aflatoxins contents at the end of storage period
might be due to the fact that with increase in storage period mould attained more resistance
against antifungal constituents of plants and proliferated to produce aflatoxins. Our findings are
in close agreement to Thanaboripat (2011) who reported the decrease in inhibitory effect of
plants with time because the active compounds in leaves became less effective.
Although aflatoxin contents increased up to 7th month in treated and control samples,
after this decrease in aflatoxin contents was observed at 9th month of storage period. This decline
in aflatoxins amount might be due to the degradation of aflatoxins because as the fungal mycelia
get matured; intra-mycelial substances are released from mycelia which may cause degradation
of aflatoxins. A similar trend of aflatoxins accumulation and degradation was also observed by
Bresler et al. (1998) in stored amaranth grains inoculated with A. parasiticus at 25 oC for 56 days
(approximately 2 months).
A variation in fungicidal potential of two plants against the toxigenic A. parasiticus strain
might be due to substantial variation in their phyto constituents (Cavaliere et al., 2006). In
present study, the maximum inhibitory effect (100%) caused by neem leaves (20%
concentration) against aflatoxins synthesis by A. parasiticus in three cereals relates with the
findings of (Zeringue and Bhatnagar, 1994) who reported that the aqueous extract of neem leaves
effectively reduced 98% aflatoxin B1 production by A. flavus in cotton balls without reducing
fungal growth, and Costa et al. (2010) also reported that aqueous extract of neem leaves at 5%

9
concentration inhibited aflatoxin B1 production up to 90% by A. parasiticus in a submerged
culture.

Conclusion
Neem at 20% fully inhibited the production of all aflatoxins for four months in wheat,
two months in maize. Kikar leaves inhibited comparatively to lesser extent. Increasing the
leaves concentration, there was a linear trend in aflatoxins inhibition for few months, however,
tests with higher leaves-concentration didn’t produce any better results as with 20%. A.
parasiticus might get resistance against inhibitory-compounds in leaves, there may be a decrease
in concentration of compounds with time. Neem leaves can be introduced in agriculture market
as a natural, safe and cheaper additive material to inhibit aflatoxins production during the storage
of cereals and animal feeds.

Acknowledgements
The authors are grateful to the Higher Education Commission for financial support
through a research project ‘‘Protection of Cereal Grains against Aflatoxins Contamination Using
Plant Materials.’’

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Distribution of Fungi and aflatoxins in stored peanut variety. Food chem. 106, 285-190.

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27. Naseer, R., Sultana, B., Khan, M.Z., Naseer, D., Nigam, P., 2014. Utilization of waste
fruit-peels to inhibit aflatoxins synthesis by Aspergillus flavus: A biotreatment of rice
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13
Table 1: Amount of aflatoxins (ng/g) produced by A.parasiticus in wheat stabilized by neem
leaves.
Storage Leaves B1A B2B G1 A G2 B
period
concentration
(months)
added (%)

Control 8.19±0.29d 0.96±0.04d 3.00±0.09d 0.65±0.03d

1 5 1.64±0.06a(80) < LOD 0.12±0.00a(96) < LOD
10 0.90±0.03a(89) < LOD < LOD < LOD
20 < LOD < LOD < LOD < LOD
Control 18.69±0.65d 2.14±0.08d 5.79±0.17d 1.24±0.05d
2 5 3.55±0.12a(81) 0±0a(100) 0.23±0.01a(96) 0±0a(100)
10 2.06±0.07a(89) 0±0a(100) 0±0a(100) 0±0a(100)
20 < LOD < LOD < LOD < LOD
Control 38.55±1.35d 4.36±0.17d 13.26±0.38d 1.78±0.07d
3 5 8.48±0.30a(78) 0.26±0.01a(94) 0.80±0.02a(94) 0.11±0.00a(94)
10 5.40±0.19a(86) < LOD < LOD < LOD
20 < LOD < LOD < LOD < LOD
Control 79.21±2.77d 9.57±0.36d 22.16±0.64d 3.19±0.13d
4 5 19.8±0.69ab(75) 0.77±0.03a(92) 2.22±0.06a(92) 0.19±0.01a(94)
10 14.26±0.50a(82) < LOD < LOD < LOD
20 < LOD < LOD < LOD < LOD
Control 98.65±3.45d 9.57±0.36d 28.16±0.82d 7.43±0.30d
5 5 31.57±1.10b(68) 1.05±0.04a(89) 3.66±0.11a(87) 0.74±0.03a(90)
10 23.68±0.83a(76) 0.38±0.01a(96) 1.23±0.04a(96) < LOD
20 8.88±0.31a(91) < LOD < LOD < LOD
Control 77.46±2.71d 7.56±0.29d 31.72±0.92d 0.20±0.01d
7 5 30.98±1.08b(60) 1.44±0.05a(81) 6.34±0.18a(80) < LOD
10 21.69±0.76a(72) 0.45±0.02a(94) 4.12±0.12a(87) < LOD

14
 20 10.84±0.38a(86) < LOD 0.95±0.03a(97) < LOD
d d
Control 29.22±1.02 3.17±0.12 17.46±0.51d 4.79±0.19d
b a a
9 5 14.02±0.49 (52) 0.85±0.03 (73) 4.71±0.14 (73) 1.20±0.05a(75)
a a a
10 11.39±0.40 (61) 0.48±0.02 (85) 3.84±0.11 (78) 0.72±0.03a(85)
a a a
20 7.01±0.25 (76) 0.13±0.00 (96) 1.75±0.05 (90) 0.19±0.01a(96)
Values are mean ± SD of each samples analyzed individually in triplicate.
Values followed by the same small letter in superscript within the same line are not significantly different (p > 0.05)
Different capital letter in superscript within the same line significantly different (p < 0.05)
Values in brackets represent percent inhibition in aflatoxins production by plant leaves

Table 2: Amount of aflatoxins (ng/g) produced by A.parasiticus in wheat stabilized by kikar

leaves.

Storage Leaves B1A B2B G1 A G2 B

period
concentratio
(months)
n added (%)

Control 8.19±0.29d 0.96±0.04d 3.00±0.09d 0.65±0.03d

1 5 3.77±0.13b(54) 0.27±0.01b(72) 0.72±0.02a(76) < LOD
10 3.36±0.12b(59) 0.10±0.00a(90) 0.3±0.01a(90) < LOD
20 2.05±0.07b(75) < LOD < LOD < LOD
Control 18.69±0.65d 2.14±0.08d 5.79±0.17d 1.24±0.05d
2 5 8.22±0.29b(56) 0.58±0.02b(73) 1.45±0.04b(75) 0.02±0.00a(98)
10 7.66±0.27b(59) 0.21±0.01a(90) 0.58±0.02a(90) < LOD
20 4.67±0.16ab(75) < LOD < LOD < LOD
control 38.55±1.35d 4.36±0.17d 13.26±0.38d 1.78±0.07d
3 5 18.89±0.66b(75) 1.26±0.05b(71) 3.84±0.11b(71) 0.05±0a(97)
10 16.96±0.59b(56) 0.44±0.02a(90) 1.59±0.05a(88) < LOD
20 9.64±0.34b(75) < LOD < LOD < LOD
control 79.21±2.77d 9.57±0.36d 22.16±0.64d 3.19±0.13d
4 5 41.19±1.44c(48) 3.06±0.12b(68) 7.54±0.22b(68) 0.26±0.01a(92)
10 38.02±1.33b(52) 1.34±0.05a(86) 3.32±0.10a(86) < LOD
20 22.97±0.80b(71) 0.29±0.01a(97) 0.66±0.02a(97) < LOD
control 98.65±3.45d 9.57±0.36d 28.16±0.82d 7.43±0.30d
5 5 60.18±2.11c(66) 3.64±0.14b(62) 10.7±0.31b(62) 0.89±0.04a(88)
10 52.29±1.83b(47) 1.91±0.07a(80) 5.35±0.16a(81) 0.37±0.01a(95)
20 33.54±1.17b(66) 0.96±0.04a(90) 2.82±0.08a(90) < LOD
control 77.46±2.71d 7.56±0.29d 31.72±0.92d 0.20±0.01d
7 5 55.00±1.92c(29) 3.40±0.13b(55) 15.23±0.44b(52) 0.02±0.00a(100)
10 47.25±1.65c(39) 2.04±0.08b(73) 8.25±0.24a(74) < LOD

15
 20 4.08±0.14a(56) 1.36±0.05a(82) 6.34±0.18a(80) < LOD
d d
control 29.22±1.02 3.17±0.12 17.46±0.51d 4.79±0.19d
c c b
9 5 23.37±0.82 (20) 1.67±0.06 (47) 9.25±0.27 (47) 1.44±0.06b(70)
c b b
10 19.87±0.70 (32) 1.20±0.05 (62) 6.11±0.18 (65) 1.01±0.04a(79)
b a a
20 15.48±0.54 (47) 0.85±0.03 (73) 4.71±0.14 (73) 0.57±0.02a(80)
Values are mean ± SD of each samples analyzed individually in triplicate.
Values followed by the same small letter in superscript within the same line are not significantly different (p > 0.05)
Different capital letter in superscript within the same line significantly different (p < 0.05)
Values in brackets represent percent inhibition in aflatoxins production by leaves

Table 3: Amount of aflatoxins (ng/g) produced by A.parasiticus in maize stabilized by neem

leaves.

Storage Leaves B1A B2B G1 A G2 B

period
concentratio
(months)
n added (%)

Control 12.17±0.43d 1.93±0.07d 4.82±0.14d 0.11±0d

1 5 3.77±0.13a (69) 0.25±0.01a(87) 1.35±0.04a (72) 0.01±0a(90)
10 2.31±0.08a(81) < LOD < LOD < LOD
20 < LOD < LOD < LOD < LOD
Control 27.7±0.97d 4.72±0.18d 8.26±0.24d 1.23±0.05d
2 5 8.59±0.3a (69) 0.57±0.02a (88) 0.41±0.01a (95) 0±0(100)
10 3.6±0.13a(87) < LOD < LOD < LOD
20 < LOD < LOD < LOD < LOD
Control 73.67±2.58d 6.82±0.26d 19.05±0.55d 2.16±0.09d
3 5 22.84±0.8b(69) 1.91±0.07b(72) 0.95±0.03a(95) 0±0(100)
10 10.31±0.36a(86) 0.27±0.01a(96) < LOD < LOD
20 0.74±0.03a(99) < LOD < LOD < LOD
Control 159.7±5.59d 14.38±0.55d 43.37±1.26d 6.52±0.26d
4 5 63.88±2.24b(60) 4.06±0.15a(72) 6.51±0.19a(85) 0.65±0.03a(90)
10 27.15±0.95a(83) 0.72±0.03a(95) 3.47±0.1a(92) 0.46±0.02a(93)
20 1.6±0.06a(99) < LOD 0.87±0.03a(98) < LOD
Control 151.25±5.29d 18.91±0.72d 45.51±1.32d 13.65±0.55d
5 5 63.54±2.22b(58) 5.86±0.22b(69) 8.19±0.24a(82) 2.59±0.10a(81)
10 40.85±1.43a(73) 1.89±0.07a(90) 3.19±0.09a(93) 1.77±0.07a(87)
20 25.67±0.9a(87) 1.89±0.07a(90) 1.37±0.04a(97) 0.55±0.02a(96)
Control 79.19±2.77d 9.23±0.35d 30.73±0.89d 10.07±0.4d
7 5 46.72±1.64b (41) 3.42±0.13b (63) 6.76±0.2a (78) 2.22±0.09a (78)
10 31.49±1.1b (64) 1.75±0.07a (81) 2.46±0.07a (92) 1.61±0.06a (84)

16
 20 15.84±0.55a (80) 0.74±0.03a (92) 3.38±0.1a (89) 0.4±0.02a (96)
d d d
Control 32.11±1.12 3.17±0.12 19.69±0.57 4.14±0.17d
b b a
9 5 19.59±0.69 (39) 1.17±0.04 (63) 4.92±0.14 (75) 0.91±0.04a(78)
a a
10 12.89±0.45(60) 0.63±0.02 (80) 1.97±0.06 (90) 0.7±0.03a(83)
a a
20 8.34±0.29(74) 0.25±0.01 (92) 2.95±0.09 (85) 0.33±0.01a(92)
Values are mean ± SD of each samples analyzed individually in triplicate.
Values followed by the same small letter in superscript within the same line are not significantly different (p > 0.05)
Different capital letter in superscript within the same line significantly different (p < 0.05)
Values in brackets represent percent inhibition in aflatoxins production by leaves

Table 4: Amount of aflatoxins (ng/g) produced by A.parasiticus in maize stabilized by kikar

leaves.

Storage Leaves B1A B2B G1 A G2 B

period
concentratio
(months)
n added (%)

Control 12.17±0.43d 1.93±0.07d 4.82±0.14d 0.11±0d

1 5 6.09±0.21b(50) 0.85±0.03b (56) 1.54±0.04b (68) 0.03±0a(77)
10 5.23±0.18b(57) 0.67±0.03a(65) 1.4±0.04a(71) 0.01±0a (90)
20 2.56±0.09a(79) 0.09±0a (100) < LOD < LOD
Control 27.7±0.97d 4.72±0.18d 8.26±0.24d 1.23±0.05d
2 5 13.85±0.48b (50) 1.94±0.07b (98.01) 2.23±0.06a (73) 0.26±0.01a (79)
10 10.25±0.36b (63) 1.32±0.05b (72.02) 0.83±0.02a (90) 0.16±0.01a (87)
20 3.32±0.12a (88) 0.25±0.01a (89) 0.29±0.01a(84) < LOD
Control 73.67±2.58d 6.82±0.26d 19.05±0.55d 2.16±0.09d
3 5 41.26±1.44c(44) 3.34±0.13b(51) 6.1±0.18b(68) 0.54±0.02a(75)
10 29.47±1.03b(60) 1.91±0.07a(72) 2.48±0.07a(87) 0.28±0.01a(87)
20 14±0.49a(81) 0.55±0.02a(92) 1.14±0.03a(94) < LOD
Control 159.7±5.59d 14.38±0.55d 43.37±1.26d 6.52±0.26d
4 5 103.81±3.63c(35) 7.19±0.27b(50) 17.35±0.5b(60) 1.89±0.08b(71)
10 70.27±2.46b(56) 5.46±0.21b(62) 11.28±0.33a(74) 1.17±0.05a(82)
20 35.13±1.23a(78) 2.16±0.08a(85) 5.2±0.15a(88) < LOD
Control 151.25±5.29d 18.91±0.72d 45.51±1.32d 13.65±0.55d
5 5 102.87±3.6c(32) 10.02±0.38b(47) 16.84±0.49b(63) 5.32±0.21b(61)
10 81.69±2.86b(46) 6.24±0.24b(67) 15.02±0.44b(67) 3.14±0.13a(77)
20 55.97±1.96b(63) 4.54±0.17a(76) 9.56±0.28a(79) < LOD
Control 79.19±2.77d 9.23±0.35d 30.73±0.89d 10.07±0.4d
7 5 62.56±2.19c (21) 5.44±0.21a (41) 14.75±0.43b (52) 4.53±0.18b (74.01)

17
 10 49.1±1.72c (38) 3.23±0.12b (65) 13.21±0.38b (57) 3.02±0.12b (70.01)
b a b
20 34.05±1.19 (57) 2.68±0.1 (71) 9.83±0.29 (68) 2.05a(85)
d d d
Control 32.11±1.12 3.17±0.12 19.69±0.57 4.14±0.17d
c d b
9 5 25.36±0.89 (21) 3.9±0.15 (37) 9.85±0.29 (50) 1.53±0.06b (63)
10 c
20.87±0.73 (35) b
1.27±0.05 (60) b
9.06±0.26 (54) 1.49±0.06b (64)
b b b
20 15.73±0.55 (51) 1.14±0.04 (64) 6.69±0.19 (66) 1.28± b(69)
Values are mean ± SD of each samples analyzed individually in triplicate.
Values followed by the same small letter in superscript within the same line are not significantly different (p > 0.05)
Different capital letter in superscript within the same line significantly different (p < 0.05)
Values in brackets represent percent inhibition in aflatoxins production by leaves

Table 5: Amount of aflatoxins (ng/g) produced by A.parasiticus in rice stabilized by neem leaves.

Storage Leaves B1A B2B G1 A G2 B

period
concentratio
(months)
n added (%)

Control 19.12±0.67d 2.72±0.10d 6.88±0.20d 0.92±0.04d

1 5 5.74±0.20b(70) 0.41±0.02a(85) 1.24±0.04a(82) 0.10±0.00a(89)
10 3.82±0.13a(80) 0.11±0.00a(96) 0.34±0.01a(95) < LOD
20 0.76±0.03a(96) < LOD < LOD < LOD
Control 44.24±1.55d 7.31±0.28d 15.04±0.44d 2.13±0.09d
2 5 15.49±0.54b(65) 1.32±0.05a(82) 3.16±0.09a(79) 3.16±0.13d(87)
10 11.95±0.42a(73) 0.58±0.02a(92) 1.05±0.03a(93) 0.02±0a(99)
20 3.98±0.14a(91) < LOD < LOD < LOD
Control 96.13±3.36d 12.43±0.47d 28.03±0.81d 3.87±0.15d
3 5 39.41±1.38b(59) 2.49±0.09a(80) 7.29±0.21b(74) 0.58±0.02a(85)
10 29.8±1.04a(69) 1.37±0.05a(89) 4.04±0.12a(87) 0.15±0.01a(96)
20 14.42±0.5a(85) < LOD < LOD < LOD
Control 178.13±6.23d 22.68±0.86d 55.31±1.6d 9.21±0.37d
4 5 87.28±3.05b(51) 5.22±0.2a(77) 16.59±0.48a(70) 1.75±0.07a(81)
10 57.91±2.03a(63) 2.95±0.11a(87) 9.96±0.29a(82) 0.64±0.03a(93)
20 35.63±1.25a(80) 1.81±0.07a(92) < LOD < LOD
Control 161.15±5.64d 22.22±0.84d 64.32±1.87d 14.47±0.58d
5 5 82.18±2.88b(49) 5.55±0.21a(75) 22.51±0.65b(65) 3.18±0.13a(78)
10 64.46±2.26a(60) 3.11±0.12a(86) 14.15±0.41a(78) 1.74±0.07a(88)
20 38.68±1.35a(76) 1.78±0.07a(92) 4.42±0.13a(92) < LOD
Control 94.49±3.31d 31.1±1.18d 66.17±1.92d 14.57±0.58d

18
 7 5 52.91±1.85b(44) 13.06±0.5b(58 25.81±0.75b(61) 3.79±0.15b(74)
ab a a
10 43.46±1.52 (54) 9.64±0.37 (69) 18.53±0.54 (72) 2.04±0.08a(86)
a a a
20 26.46±0.93 (72) 4.35±0.17 (86) 11.25±0.33 (83) 0.58±0.02a(96)
d d d
Control 56.19±1.97 19.3±0.73 41.11±1.19 9.34±0.37d
b a b
9 5 35.4±1.24 (37) 7.53±0.29 (61) 18.09±0.52 (56) 2.62±0.1b(72)
ab b a
10 29.22±1.02 (48) 9.46±0.36 (51) 12.33±0.36 (70) 1.77±0.07a(81)
a a a
20 20.23±0.71 (64) 4.05±0.15 (79) 8.63±0.25 (79) 0.93±0.04a(90)
Values are mean ± SD of each samples analyzed individually in triplicate.
Values followed by the same small letter in superscript within the same line are not significantly different (p > 0.05)
Different capital letter in superscript within the same line significantly different (p < 0.05)
Values in brackets represent percent inhibition in aflatoxins production by leaves

Table 6: Amount of aflatoxins (ng/g) produced by A.parasiticus in rice stabilized with kikar leaves.

Storage Leaves B1A B2B G1 A G2 B

period
concentration
(months)
added (%)

Control 19.12±0.67d 2.72±0.10d 6.88±0.20d 0.92±0.04d

1 5 7.84±0.27b(59) 0.84±0.03b(69) 1.99±0.06b(71) 0.14±0.01a(85)
10 6.69±0.23b(65) 0.60±0.02a(78) 1.65±0.05a(76) < LOD
20 5.16±0.18a(73) 0.38±0.01a(86) 0.55±0.02a(92) < LOD
Control 44.24±1.55d 7.31±0.28d 15.04±0.44d 2.13±0.09d
2 5 22.12±0.77b(50) 2.56±0.1b(65) 4.66±0.14b(69) 0.32±0.01a(85)
10 18.58±0.65b(58) 1.75±0.07a(76) 4.21±0.12b(72) 0.09±0a(96)
20 13.27±0.46a(70) 1.1±0.04a(85) 1.5±0.04a(90) < LOD
Control 96.13±3.36d 12.43±0.47d 28.03±0.81d 3.87±0.15d
3 5 56.72±1.99c(41) 4.85±0.18b(61) 10.09±0.29b(64) 0.7±0.03a(82)
10 45.18±1.58b(53) 3.35±0.13a(73) 8.97±0.26b(68) 0.35±0.01a(91)
20 35.57±1.24b(63) 2.24±0.08a(82) 3.92±0.11a(86) < LOD
Control 178.13±6.23d 22.68±0.86d 55.31±1.6d 9.21±0.37d
4 5 121.13±4.24c(32) 9.3±0.35b(59) 22.12±0.64a(60) 2.12±0.08a(77)
10 106.88±3.74bc(40) 7.26±0.28b(68) 17.7±0.51a(68) 1.29±0.05a(86)
20 78.38±2.74b(56) 4.54±0.17a(80) 11.06±0.32a(80) 0.28±0.01a(97)
Control 161.15±5.64d 22.22±0.84d 64.32±1.87d 14.47±0.58d
5 5 112.8±3.95c(30) 9.33±0.35b(58) 28.94±0.84b(55) 4.05±0.16b(72)
10 96.69±3.38b(40) 7.11±0.27b(68) 25.73±0.75b(60) 2.6±0.1a(82)
20 75.74±2.65b(53) 4.44±0.17a(80) 16.72±0.48b(74) 0.87±0.03a(94)

19
 Control 94.49±3.31d 31.1±1.18d 66.17±1.92d 14.57±0.58d
c b b
7 5 70.86±2.48 (25) 15.55±0.59 (50) 34.41±1.00 (48) 5.24±0.21b(64)
c b b
10 61.42±2.15 (35) 12.75±0.48 (59) 29.78±0.86 (55) 4.08±0.16b(72)
b a a
20 51.97±1.82 (45) 9.33±0.35 (70) 22.5±0.65 (66) 1.89±0.08a(87)
d d d
Control 56.19±1.97 19.3±0.73 41.11±1.19 9.34±0.37d
c c b
9 5 45.52±1.59 (19) 11.19±0.43 (42) 23.43±0.68 (43) 3.74±0.15b(60)
c b b
10 41.02±1.44 (27) 9.65±0.37 (50) 20.56±0.6 (50) 2.99±0.12b(68)
b b ab
20 35.40±1.24 (37) 7.91±0.3 (59) 16.86±0.49 (59) 1.68±0.07a(82)
Values are mean ± SD of each samples analyzed individually in triplicate.
Values followed by the same small letter in superscript within the same line are not significantly different (p > 0.05)
Different capital letter in superscript within the same line significantly different (p < 0.05)
Values in brackets represent percent inhibition in aflatoxins production by leaves

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HIGHLIGHTS
_ Aspergillus parasiticus grew and produced aflatoxins in cereals (rice, maize
and wheat) on storage.
_ Neem and kiker leaves replaced chemical treatments to inhibit aflatoxin production.
_ Utilisation of waste leaves is economical and safer for long-term storage of cereals.
_ Saving cereals of economic value by using waste leaves of two plants

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